In the fields of biotechnology, biomedicine, and bioremediation, for example, bioactive species are often immobilized onto a support member to more effectively utilize the bioactive species. The term "immobilize," and its derivatives, as used herein refers to the attachment of a bioactive species directly to a support member or to a support member through at least one intermediate component. As used herein, the term "attach" and its derivatives refer to adsorption, such as, physisorption or chemisorption, ligand/receptor interaction, covalent bonding, hydrogen bonding, or ionic bonding of a polymeric substance or a bioactive species to a support member. Bioactive species include enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, carbohydrates, oleophobics, lipids, pharmaceuticals, and therapeutics, for example. Cells, such as, mammalian cells, reptilian cells, amphibian cells, avian cells, insect cells, planktonic cells, cells from non-mammalian marine vertebrates and invertebrates, plant cells, microbial cells, protists, genetically engineered cells, and organelles, such as mitochondria, are also bioactive species. In addition, non-cellular biological entities, such as viruses, virenos, and prions are considered bioactive species.
There are various materials suitable for use as a support member for immobilizing bioactive species. Examples of these materials include hydrocarbon polymers, fluorocarbon polymers, ceramics, and metals. Of these materials, polytetrafluoroethylene and porous polytetrafluoroethylene, are of particular interest as support members. Polytetrafluoroethylene (PTFE) is a hydrophobic fluorocarbon polymer well known to have exceptional resistance to solvent and chemical attack. Porous polytetrafluoroethylene can be made in a variety of ways. For example, coextrusion of a polytetrafluoroethylene extrudate with a readily vaporizable material, such as naptha, forms a material from which the readily vaporizable material is ultimately removed to render the material porous (See U.S. Pat. No. 3,281,511, which is incorporated herein by reference). Expanded polytetrafluoroethylene (ePTFE) or stretched polytetrafluoroethylene are porous fluorocarbon polymer materials characterized primarily by a multiplicity of interconnecting voids defined by nodes and fibrils of polytetrafluorethylene material. Expanded PTFE materials, including ePTFE membranes and films described hereinbelow, may be made according to the teachings of U.S. Pat. Nos. 3,953,566, 3,962,153, 4,096,227, 4,187,390, and 4,902,423, each of which is incorporated herein by reference. In general, porous PTFE materials are chemically stable and very biocompatible. The materials have easily controlled pore sizes with large surface area/volume ratios, good mechanical strength, and good resistance to fouling, among other properties. Accordingly, these materials are attractive as support members for the immobilization of bioactive species.
Several methods for immobilizing bioactive species onto fluorocarbon polymers, such as PTFE, porous PTFE, or hydrocarbon polymeric materials have been taught in the literature. These methods include, for example, chemical modifications of the polymeric material to form chemically reactive groups thereon for covalent bonding of a bioactive species to the material, adsorption of a bioactive species to the polymeric material, and modifying the polymeric material and/or a bioactive species with compatibilizers, surfactants, or wetting agents to alter their surface energies. As described below, there are difficulties with each of these methods.
Due to the extreme chemical inertness of the backbone of fluorocarbon polymers and many hydrocarbon polymers, only highly energetic classes of reactions can successfully alter the backbone of these materials to produce chemically reactive organic moieties thereon. When chemically reactive organic moieties are formed along the backbone of a fluorocarbon or hydrocarbon polymer, bioactive species can be attached to the moieties. For example, enzymes have been chemically attached onto ammonia plasma-treated fluoropolymer surfaces using ammonia plasma and glutaraldehyde (See M. Kawakami, H. Koya, and S. Gondo, "Immobilization of glucose oxidase on polymer membranes treated by low-temperature plasma," Biotech. Bioeng., 32: 369, (1988)), or aminosilane and glutaraldehyde (J. M. Taylor, M. Cheryan, T. Richardson, and N. F. Olson, "Pepsin immobilized on inorganic supports for the continuous coagulation of skim milk," Biotech. Bioeng., 19: 683 (1977), for example). Representative examples of these highly energetic reactions include, thermal decomposition (U.S. Pat. No. 5,296,510, issued to Shigeru et al.), irradiation with electrons, gamma rays, radio waves, microwaves (T. Kasemura, S. Ozawa, K. Hattori, "Surface Modification of Fluorinated Polymers by Microwave Plasmas," J. Adhesion, 33: 33 (1990); Commonwealth Scientific and Industrial Research Organisation in PCT/AU89/00220; Y. Ito, Y. Iguchi, T. Kashiwagi, Y. Imanisihi, "Synthesis and nonthrombogenicity of polyetherurethaneurea film grafted with poly(sodium vinyl sulfonate)," J. Biomed. Mater. Res., 25: 1347 (1991); and Y. Ito, M. Kajihara, Y. Imanishi, "Materials for enhancing cell adhesion by immobilization of cell adhesive peptide," J. Biomed. Mater. Res., 25: 1325 (1991)), or UV light (K. Allmer, A. E. Feiring, "Photochemical Modification of a Fluoropolymer Surface," Macromolecules, 24: 5487 (1991); A. B. Pointer, W. R. Jones Jr., R. H. Janse, "Surface energy changes produced by ultraviolet-ozone irradiation of poly(methyl methacrylate), polycarbonate, and polytetrafluoroethylene," Polym. Eng. Sci., 34: 1233 (1994)), glow discharge irradiation (R. Sipehia, G. Martucci, M. Barbarosie, C. Wu, "Enhanced Attachment of and Growth of Human Endothelial Cells Derived from Umbilical Veins on Ammonia Plasma Modified Surfaces of PTFE and ePTFE Synthetic Vascular Graft Biomaterials," Biomat. Art. Cells Immob. Biotech., 21:455 (1993)), and redox reactions with strong nucleophiles such as sodium or potassium aryloxides (C. A. Costello, T. J. McCarthy, "Surface-selective Introduction of Specific Functionalities onto Poly(tetrafluoroethylene)," Macromolecules, 20: 2819 (1987); A. J. Dias, T. J. McCarthy, "Introduction of carboxylic acid, aldehyde, and alcohol functional groups onto the surface of poly(chlorotrifluoroethylene)," Macromolecules, 20: 2068 (1987); G. E. Sweet, J. P. Bell, "Selective chemical etching of poly(ethylene terephthalate) using primary amines," J. Polym. Sci. Phys. Ed., 16: 1935 (1978); and H. B. Lin, S. L. Cooper, "Polyurethane copolymers containing covalently attached RGD-peptide," Mat. Res. Soc. Symp. Proc., 252: 185 (1992)), or ozone (R. L. Daubendiek, J. G. Calvert, "The Reaction of Ozone with Perfluorinated Polyolefins," Environ. Lett., 6: 253 (1974)).
These high-energy modifications can be highly destructive to polymeric materials, however. With PTFE and porous PTFE, for example, high-energy modifications of the fluorocarbon backbone often leads to uncontrolled surface erosion of the material, depolymerization of the perfluorocarbon backbone (A. B. Pointer, W. R. Jones Jr., R. H. Jansen, "Surface Energy Changes Produced by Ultraviolet-ozone Irradiation of Poly(methyl methacrylate), Polycarbonate, and Polytetrafluoroethylene," Polym. Eng. Sci., 34: 1233 (1994)), reduction in the strength of the polymer substrate (S. Kanazawa, T. Takiguchi, A. Nishimora, T. Morita, and A. Uno, "Development of a Hydrophilic PTFE porous membrane filter," Sumitomo Denki; 147:99 (Sept., 1995), and loss of defined fibrillar structure of porous expanded PTFE (U.S. Pat. No. 5,462,781, issued to Zukowski).
Furthermore, high energy modifications to hydrocarbon polymers and fluorocarbon polymers, such as PTFE or porous PTFE, often produce reactive compositions on the hydrocarbon or fluorocarbon backbone that have an undeterminable surface density, chemical identity, and chemical stability (X. Xie, T. R. Gengenbach, and H. J. Griesser, "Changes in wettability with time of plasma modified perfluorinated polymers," J. Adhesion Sci. Technol., 6:1411 (1992)). In addition, the modification of the hydrocarbon or perfluorocarbon backbone may be spatially uneven, with microscopic or macroscopic areas of the polymer remaining unmodified. At best, these surface compositions can only be empirically determined and so may be only partially known.
With a hydrophobic porous polymer, such as ePTFE or porous polyethylene, for example, modification of the backbone may be limited to the outer layers of the porous material, with the inner pore structures remaining mostly or completely unmodified. As a result, the unmodified regions of the polymer support member remain hydrophobic and so do not readily support wetting with high surface tension fluids. In the absence of such wetting, continuous passage of high surface tension fluids through the support member for mass transport of reactants or nutrients to and from an immobilized bioactive species cannot be established or maintained. Intermittent or incomplete passage of high surface tension fluid phases in a porous polymer support member can lead to channeling of the high surface tension fluid through only portions of the porous material, resulting in reduced efficiency. Moreover, these unmodified areas cannot be immobilized with bioactive species, resulting in inefficient use of the high surface area of the porous material.
Due to these limitations, modification of the backbone of a hydrocarbon or fluorocarbon polymer support member with a high energy reaction is most often an unsuitable method for immobilization of bioactive species.
As an alternative to these chemical modifications of polymeric surfaces, nonchemical methods have been employed to attach bioactive species to hydrocarbon and fluorocarbon support members. In the simplest method, a bioactive species is immobilized onto the surfaces of a fluorocarbon polymer via simple physicochemical adsorption. For example, M. Rucha, B. Turkiewicz, and J. S. Zuk, "Polymeric membranes for lipase immobilization," J. Am. Oil Chem. Soc., 67: 887 (1990) and Shults, M. et al., "Continuous In Vivo Glucose Analysis Based On Immobilized Enzyme Bound To Derivatized Teflon Membrane," Trans. ASAIO, 25: 66 (1979) each teach enzyme physisorption onto ePTFE. However, physisorption of bioactive species is often kinetically and thermodynamically unstable, highly reversible, and competitively displaced by solution phase reactants, products, or nutrients. In addition, physisorption may alter or damage the bioactive species. Thus, physisorption of bioactive species to hydrocarbon polymer or fluorocarbon polymer support members is not usually a suitable immobilization technique. Furthermore, the hydrophobic properties of a porous hydrocarbon polymer or porous fluorocarbon polymer support member, such as ePTFE, often prevent physisorption of a bioactive species in the inner pore structures of the support member.
In another non-chemical immobilization scheme, bioactive species such as cells have been immobilized to porous PTFE support members. For example, bacteria and yeast cells have been immobilized in PTFE fibril matrices using an emulsion of PTFE and surfactants (F. W. Hyde, G. R. Hunt, and L. A. Errede, "Immobilization of bacteria and Saccharomyces cerevisiae in poly(tetrafluoroethylene) membranes," Appl. Environ. Microbial., 57: 219 (1991)) The surfactant is necessary to ensure for the presence of a continuous water phase throughout the emulsion in order to allow diffusion of nutrients to cells immobilized within the porous regions of the material. This method for immobilization of cells in the porous matrices of a fluorocarbon polymer is quite harsh, however, as many surfactants are cytotoxic. As a result, this method is not generally applicable to all cell types because the reaction conditions are often toxic to cell types such as plant or mammalian cells. In addition, the surfactant may initially obstruct some or all of the void space of the pores in such a porous support member. Lastly, the surfactant may leach from the support member with time. This often presents a toxic environment to the immobilized cells or channelling of nutrients, resulting in a heterogeneous distribution of cells within the porous support member. Accordingly, this is often an unsuitable method for the immobilization of bioactive species.
In yet another non-chemical method, mammalian cells have been immobilized within ePTFE support members by forcing the cells into the pores of the material by hydrostatic pressure (University of Washington, PCT/US95/03735). However, cell viability is often low due to mechanical shearing forces produced during the process. In addition, the ePTFE remains hydrophobic and mass transport of liquid water across the thickness of the material may remain low, resulting in suboptimal transport of nutrients to the immobilized cells.
In an attempt to improve the immobilization of bioactive species adsorbed onto hydrocarbon or fluorocarbon polymer support members, the hydrophobicity of the surfaces of such polymer support members can be modified with hydrophilic surface active agents, or surfactants. Hydrophobic surfaces are low energy surfaces that are readily wetted by low surface tension fluids, such as low molecular weight hydrocarbons or alcohols, and most low molecular weight organic solvents, such as benzene, acetone, toluene, and dioxane, etc. Hydrophilic surfaces, on the other hand, are high energy surfaces that are readily wetted by high surface tension fluids. Examples of high surface tension fluids include, but are not limited to, liquid water, aqueous salt and protein solutions, dimethyl formamide, dimethyl sulfoxide, glycerol, hexamethyl phosphorictriamide, formamide, and glycol, for example. In these methods, surfactants were physisorbed onto the surfaces of a polymeric hydrocarbon or fluorocarbon support member. When a surfactant is applied to the surfaces of a hydrophobic polymeric support member, the surface energy of the support member is usually increased. The increased surface energy of the support member often facilitates attachment of a bioactive species to the support member. For example, U.S. Pat. Nos. 5,077,215, issued to McAuslan et al., 5,183,545, issued to Branca et al., and 5,203,997 issued to Koyama et al., teach the adsorption of anionic and nonionic fluorocarbon surfactants to the surface of fluorocarbon support members to modify their hydrophobicity. As a result, the normally hydrophobic surface of the polymer was rendered more hydrophilic. This was followed by physisorption of a bioactive species onto the surfactant-modified polymer surface.
In similar methods, the adsorption of surfactants onto polymeric support members is taught wherein bioactive species are subsequently bound to the adsorbed surfactants. For example, U.S. Pat. No. 5,263,992, issued to Guire et al., teaches adsorption of polymeric chains to a support member, to which biomolecules are attached through a photoactive agent. In this manner, bioactive species were more readily immobilized onto the support member than if directly immobilized onto the underlying hydrophobic polymer surface. Alternatively, the bioactive species may be covalently bonded directly to the surfactant rather than physisorbed thereto (See also, U.S. Pat. No. 4,619,897, issued to Hato et al., for example).
In these methods, the presence of a surfactant provided for a surface having a hydrophilicity that initially enhanced the immobilization of bioactive species. For porous support members, the surfactant also initially provided continuous water phases through the pores of the support member. As discussed in greater detail below, immobilization of bioactive species with surfactants is usually unstable over time, however.
The physicochemical stability of the immobilized bioactive species is another concern when bioactive species are immobilized with surfactants. The adsorption of surfactants to a polymeric support member can serve to enhance the strength of biospecies adsorption, but the biospecies may desorb from the surfactant-treated surface nevertheless. In order to improve the retention of the bioactive species on a support member, U.S. Pat. No. 4,885,250, issued to d'Eveleigh, teaches modifying biological ligands themselves with surfactants prior to physisorption of the surfactant onto the support member. However, modification of bioactive species with surfactants prior to physisorption onto hydrophobic polymer surfaces may dramatically impact the bioactivity of the bioactive species. For example, the enzyme lactate dehydrogenase was admixed with surfactants and physisorbed onto PTFE microparticles resulting in a drop in enzymatic activity of 96% (N. D. Danielson, and R. W. Siergiej, "Immobilization of enzymes on polytetrafluoroethylene particles packed in HPLC columns," Biotech. Bioeng., 23: 1913 (1981)). In another example, the enzyme urease was modified with perfluoroalkyl chains and then physisorbed onto ePTFE resulting in an initial drop in enzymatic activity of 10-18% and poor overall enzymatic stability (R. K. Kobos, J. W. Eveleigh, M. L. Stepler, B. J. Haley, S. L. Papa, "Fluorocarbon-Based Immobilization Method for Preparation of Enzyme Electrodes," Anal. Chem., 60: 1996 (1988)). Additionally, adsorbed surfactant-modified bioactive species may remain molecularly motile and may migrate and cluster on the surface of a hydrocarbon polymer or fluorocarbon polymer support member. This effect may be accelerated with the application of cosolvents, changes in pH, or elevated temperatures, such as autoclaving, often leading to significant reorganization of the bioactive species on the surface of the support member. This can cause a loss in wetting potential of the support member with high surface tension liquids and/or uneven spatial immobilization of a bioactive species (U.S. Pat. No. 5,352,511, issued to Abaysekara et al.). As a result of these limitations, modification of a bioactive species with a surfactant prior to immobilizing the bioactive species to a hydrophobic support member is most often an unsuitable method.
Surfactants, whether physisorbed onto a hydrophobic support member or attached to a bioactive species and then adsorbed to a hydrophobic support member, are subject to desorption. For example, R. I. Foster, et al. in "Analysis of Urokinase Immobilization on the polytetrafluoroethylene vascular prosthesis," Am. J. Surg., 156: 130 (1988) teach adsorption of the hydrocarbon surfactant tridodecylmethyl-ammonium chloride onto the surfaces of ePTFE followed by immobilization of the enzyme urokinase to the surfactant. The adsorption was unstable and the enzyme/surfactant construct was eventually displaced. An additional limitation with this immobilization method is the tendency for the surfactant to leach into the solute phase, often with undesired consequences, such as contamination or inactivation of desired products or the bioactive species. Accordingly, immobilization of bioactive species onto a hydrophobic support member with a surfactant is usually unstable, short-lived, and potentially harmful to the bioactive species.
The stability of surfactant adsorption on a hydrocarbon or fluorocarbon polymer surface can be enhanced by increasing the molecular weight of the surfactant (J. H. Lee, J. Kopecek, J. D. Andrade, "Protein-Resistant Surfaces Prepared by PEO-Containing Block Copolymer Surfactants," J. Biomed. Mater. Res., 23: 351 (1989)) or by lowering the molecular entropy of the surfactant (J. H. Lee, P. Kopeckova, J. Kopecek, J. D. Andrade, "Surface Properties of Alkyl Methacrylates with Methoxy (polyethylene oxide) Methacrylates and Their Application as Protein-resistant Coatings," Biomaterials, 11: 455 (1990)). Either of these approaches can be accomplished by producing surfactants with branched or comb-like hydrocarbon chains, rather than linear hydrocarbon chains, for example. However, the stability of the adsorbed surfactant may still be transient, albeit much stronger, and may desorb from the support surface nevertheless, rendering this technique potentially unfeasible for long term applications.
In another method to reduce surfactant desorption or surfactant motility, adsorbed surfactant chains may be covalently cross-linked to adjacent adsorbed surfactant chains, producing new surface-bound planar molecules. The resulting planar molecules are of very high molecular weight and have greatly reduced molecular entropy. This type of cross-linking dramatically reduces the incidence of desorption or surface migration of the surfactant. For example, U.S. Pat. Nos. 4,929,666, issued to Schmidt et al., and 5,006,624, issued to Schmidt et al. teach adsorbing copolymers of fluoroalkyl acrylates and carboxylic vinyls, respectively, onto fluorocarbon polymer surfaces, followed by surface cross-linking of adsorbed carboxylic moieties to produce a coating highly stable to surface reorganization or to desorption. In another example, a hydrophilic fluorocarbon polymer of tetrafluoroethylene-co-vinyl alcohol was adsorbed and chemically cross-linked to itself using a polyether bisoxirane cross-linker (U.S. Pat. No. 5,354,587, issued to Abaysekara). In another example, a hydrophilic hydrocarbon polymer of polyvinyl alcohol was adsorbed and cross-linked to itself using a dialdehyde cross-linker (U.S. Pat. Nos. 4,113,912 and 4,193,138, both issued to Okita). This resulted in the ePTFE surface being rendered wettable with liquid water and the adsorbed copolymer molecules being highly resistant to desorption or to surface migration. However, these hydrophilic surfaces have limited or greatly reduced numbers of chemically functional groups to which additional polymers or bioactive species can be attached. If the adsorbed copolymer layer is deficient in the desired number or identity of chemically functional groups it possesses, then the capacity of the adsorbed copolymer or surfactant layer to immobilize bioactive species will always remain suboptimal. Accordingly, this type of cross-linking of adsorbed surfactant or copolymer molecules on hydrocarbon or fluorocarbon polymer support members is often unsuitable for the subsequent immobilization of bioactive species.
A support member having chemically stable and chemically variable hydrophilic polymeric surfaces attached thereto as a substrate upon which bioactive species are stably and efficiently immobilized would be useful. Such a construction would enable a practitioner to increase the number and/or variety of immobilized bioactive species. A practitioner would also be able to select a conjugation scheme for immobilization of a bioactive species that is best suited for the particular bioactive species.